MicroRNAs have been discovered in a wide range of eukaryotes and at 21 to 24 nt are the smallest genes discovered in any organism. The first two miRNAs, lin-4 and let-7, emerged from the genetic analysis of the C. elegans developmental timing pathway. These miRNAs downregulate via complementary 3' UTR elements the translation of specific target mRNAs revealed by genetic analysis. The let-7 miRNA was the first shown to be conserved in animal phylogeny. Other tiny RNAs, the siRNAs, emerged from the biochemical analysis of RNA interference (RNAi). A common biochemical pathway processes both the siRNAs and miRNAs: the same Dicer RNAse and members of the PIWI class of proteins. Now hundreds of miRNAs have been discovered biochemically and by informatics, many of which are conserved across animal phylogeny. In this proposal we explore using RNAi the components of the pathways that process and present miRNAs and siRNAs to target mRNAs. We also explore the particular functions of specific miRNAs that are conserved in animal phylogeny we will fuse a number of conserved miRNA genes to GFP to reveal patterns of expression. We will observe the phenotypes of the deletion mutant strains for particular miRNA genes to explore their function. Candidate target mRNAs will be identified by bioinformatics and RNAi inactivated to test their action the pathway. We will also explore the function of miRNAs in the nervous system. We will use RNAi and genetics to discover genes that are necessary for miRNA repression of translation and siRNA induced mRNA degradation. We will test whether the proteins so identified are in complexes with miRNAs and target mRNAs. We will also genetically study the process by which RNAi is negatively regulated. We will explore whether miRNAs can act as signals between cells.